1. Field of the Invention
The present invention relates, in general, to a glycoprotein that is a member of the epidermal growth factor superfamily. In particular, the present invention relates to a DNA segment encoding the glycoprotein; to a recombinant DNA molecule containing the DNA segment; to cells containing the recombinant DNA molecule; to a method of producing the glycoprotein; and to methods of disease diagnosis and therapy that involve the use of the glycoprotein or DNA segment encoding same.
2. Background Information
Cell proliferation and differentiation are regulated by polypeptide growth factors (Gospodarowicz and Moran, 1976), which play critical roles in processes leading to both normal and abnormal development (Mercola and Stiles, 1988; Cross and Dexter, 1991). Regulation of cell behavior by growth factors or hormones usually occurs by an endocrine or paracrine mechanism, in which the factor is secreted by one cell and acts at the surface of a different cell (Sporn and Todaro, 1980). In some instances regulation may occur by an autocrine mechanism, in which the growth factor acts on the same cell by which it is secreted (Sporn and Todaro, 1980). For example, secretion of TGF-.alpha. by tumor cells with EGF receptors provides an autocrine mechanism for their stimulation (Derynck, 1988). The demonstration of membrane-associated growth factors has led to the proposal of alternative stimulation mechanisms. Massague (1990) has proposed a "juxtacrine" mechanism, an alternative version of paracrine stimulation, in which cell surface growth factor on one cell stimulates growth by interaction with a receptor on the cell surface of a second cell. This mechanism has been observed in cells transfected with cDNA for TGF-.alpha. precursor whose cleavage to a secretable product was blocked (Wong et al, 1989; Anklesario et al, 1990). An alternative, auto-stimulatory mechanism has been suggested for transformation by the v-sis oncogene (Bejcek et al, 1989). A mutant v-sis carrying a KDEL endoplasmic reticulum localization signal caused stimulation of the cells even though the growth factor produced was not secreted (Bejcek et al, 1989). This intracellular activation mechanism may also occur with the unmodified oncogene and explain the inability of added antibodies against the sis-encoded protein to block v-sis transformation and the failure of high levels of exogenous v-sis protein to induce transformation of nontransformed cells (Bejcek et al, 1989).
Although growth factors clearly contribute to cancer, the roles that they play are still unclear. PDGF/sis is a potent transforming agent when transfected into fibroblasts. EGF and TGF-.alpha. are much less effective, although some cells can be transformed by transfection and selection protocols (Rosenthal et al, 1986; Watanabe et al, 1987). However, over-expression of the EGF receptor led to an amplified growth response to EGF (Kraus et al, 1988). Thus, the oncogenic potential of growth factors may depend on forming a sufficient amount of ligand-receptor complexes in the cell membrane(s). However, there is little existing evidence for such membrane ligand-receptor complexes in tumors.
Sialomucins are large, highly glycosylated glycoproteins containing predominantly O-linked carbohydrate (Carraway et al (1986) Mol. Cell Biochem. 72:109-120). Soluble sialomucins are secreted by epithelial tissues and perform a protective function for the epithelial surfaces (Neutra et al (1987) In Physiology of the Gastrointestinal Tact (Johnson, L. R., ed.) 2nd Ed., pp. 975-1009, Raven Press, New York). A second class of sialomucins is found on the cell surfaces of many carcinomas (Carraway et al (1986) Mol. Cell Biochem. 72:109-120; Carraway et al (1991) Glycobiology 1:131-138) and are postulated to provide protection from immune destruction. Since the sialomucins are long, rod-like structures, they are presumed to extend from the cell surfaces and act as an "anti-immunorecognition" barrier for the tumor cell by masking other cell surface antigens. In support of this hypothesis, the presence of sialomucins has been shown to inhibit binding of antibodies to the histocompatibility complex in mouse TA3-Ha ascites mammary adenocarcinoma cells (Codington et al (1983) Biomembranes 11:207-258), provide resistance to killing of 13762 rat mammary adenocarcinoma cells by natural killer cells (Sherblom et al (1986) Cancer Res. 86:4543-4546; Bharathan et al (1990) Cancer Res. 50:5250-5256) and correlate with metastatic potential in 13762 sublines selected for variable metastasis (Steck et al (1983) Exp. Cell Res. 147:255-267).
As a model system for tumor sialomucin properties and expression, ascites sublines of the 13762 rat mammary adenocarcinoma have been studied in which a cell surface sialomucin designated ASGP-1 is highly overexpressed (Sherblom et al (1980) J. Biol. Chem. 255:783-790). In these cells, the sialomucin is at least 0.5% of the total cell protein (Sherblom et al (1980) J. Biol. Chem. 255:783-790). Moreover, it is indirectly associated with the plasma membrane via a 1:1 molecular complex with a second glycoprotein, ASGP-2 (Sherblom et al (1980) J. Biol. Chem. 255:12051-12059), the membrane-associated component of the complex (Sheng et al (1989) J. Cell Biochem. 40:453-466), which contains primarily N-linked oligosaccharides (Hull et al (1990) Biochem. J. 265:121-129).
Recently, it has been shown that the complex is synthesized as a high M.sub.r precursor, pSMC-1, which is cleaved to yield the two components at an early stage of its transit from the endoplasmic reticulum to the cell surface (Sheng et al (1990) J. Biol. Chem. 265:8505-8510).
The present invention is based, at least in part, on studies resulting in the isolation of cDNAs coding for the carboxyl-terminal portion of pSMC-1, including the entirety of ASGP-2. These studies were undertaken in order to molecularly characterize sialomucin complex expression, its association with the cell surface and its function. The amino acid sequence derived from this cDNA shows the possible cleavage site between the ASGP-1 and ASGP-2 molecules and correlates with the biochemical properties previously demonstrated for ASGP-2, the membrane-associated glycoprotein (Hull et al (1990) Biochem. J. 265:121-129). Surprisingly, this amino acid sequence has also been found to contain two segments with strong similarities to EGF-like domains (Davis (1990) New Biologist 2:410-419). Such domains have been found not only in the growth factors (Carpenter et al (1990) J. Biol. Chem. 265:7709-7712; Appella et al (1988) FEBS Lett. 231:1-4), but also in blood coagulation proteins (Furie et al (1988) Cell 53:505-518), cell-cell adhesion factors (Bevilacqua et al (1989) Science 243:1160-1164), extracellular matrix proteins (Engel (1989) FEBS Lett. 21:1-7) and cell lineage markers for early development (Maine et al (1990) BioEssays 12:265-271). The presence of these domains in the tumor cell surface complex indicates that they perform similar proliferative/recognition functions in these highly metastatic tumor cells.